No description available.

<p>Human Coronavirus NL-63 is a respiratory virus with a high incidence rate, causing mild respiratory infections in children under the age of 18. The outbreak of Sever Acute Respiratory&nbsp / Syndrome (SARS) in 2003 sparked increased interest into the field of coronavirology and respiratory diseases subsequently led to the discovery of this novel Human Coronavirus (HCoV) by a group of scientists in Holland. The membrane protein (M) of NL-63 has been shown to interact with the nucleocapsid, spike and envelope proteins of the virus when expressed ex vivo. In contrast, the envelope protein (E) is shown to exhibit ion channel activity, interacts with the membrane protein during the formation of viral-like particles. The functions of the open reading frame 3 (ORF3) proteins remains a mystery. Research does, however, indicate that this protein is needed for in vivo infectivity and pathogenesis. Bioinformatic analysis indicates that both the ORF3 and M proteins posses at least 3&rsquo / C-terminal transmembrane regions. To further characterize the biological activity of these three proteins in clinical and laboratory samples, sensitive and specific antibodies are required. Thus, the antigenic regions of ORF3, M and the entire E gene were amplified by PCR and ligated into a bacterial expression vector for expression and subsequent generation of antibodies in a mouse system. The identities of the cloned genes were confirmed by sequencing before being expressed in an in vitro bacterial system. Western Blots were used to identify the expression of the 41kDa, 42kDa and 34kDa GST-tagged viral proteins which were consistent with the bioinformatically predicted protein species. Verified fusion proteins were expressed in large quantities, quantified and concentrated for in vivo antibody production. Inoculation of 9 healthy, female Balb/C mice with the purified fusion proteins yielded high titers of polyclonal antibodies. Western Blotting was once again used to validate the production of the antibodies before their specificity was quantitatively measured using a modified competition ELISA.</p>

<p>Human Coronavirus NL-63 is a respiratory virus with a high incidence rate, causing mild respiratory infections in children under the age of 18. The outbreak of Sever Acute Respiratory&nbsp / Syndrome (SARS) in 2003 sparked increased interest into the field of coronavirology and respiratory diseases subsequently led to the discovery of this novel Human Coronavirus (HCoV) by a group of scientists in Holland. The membrane protein (M) of NL-63 has been shown to interact with the nucleocapsid, spike and envelope proteins of the virus when expressed ex vivo. In contrast, the envelope protein (E) is shown to exhibit ion channel activity, interacts with the membrane protein during the formation of viral-like particles. The functions of the open reading frame 3 (ORF3) proteins remains a mystery. Research does, however, indicate that this protein is needed for in vivo infectivity and pathogenesis. Bioinformatic analysis indicates that both the ORF3 and M proteins posses at least 3&rsquo / C-terminal transmembrane regions. To further characterize the biological activity of these three proteins in clinical and laboratory samples, sensitive and specific antibodies are required. Thus, the antigenic regions of ORF3, M and the entire E gene were amplified by PCR and ligated into a bacterial expression vector for expression and subsequent generation of antibodies in a mouse system. The identities of the cloned genes were confirmed by sequencing before being expressed in an in vitro bacterial system. Western Blots were used to identify the expression of the 41kDa, 42kDa and 34kDa GST-tagged viral proteins which were consistent with the bioinformatically predicted protein species. Verified fusion proteins were expressed in large quantities, quantified and concentrated for in vivo antibody production. Inoculation of 9 healthy, female Balb/C mice with the purified fusion proteins yielded high titers of polyclonal antibodies. Western Blotting was once again used to validate the production of the antibodies before their specificity was quantitatively measured using a modified competition ELISA.</p>

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Human Coronavirus NL-63 is a respiratory virus with a high incidence rate, causing mild respiratory infections in children under the age of 18. The outbreak of Sever Acute Respiratory Syndrome (SARS) in 2003 sparked increased interest into the field of coronavirology and respiratory diseases subsequently led to the discovery of this novel Human Coronavirus (HCoV) by a group of scientists in Holland. The membrane protein (M) of NL-63 has been shown to interact with the nucleocapsid, spike and envelope proteins of the virus when expressed ex vivo. In contrast, the envelope protein (E) is shown to exhibit ion channel activity, interacts with the membrane protein during the formation of viral-like particles. The functions of the open reading frame 3 (ORF3) proteins remains a mystery. Research does, however, indicate that this protein is needed for in vivo infectivity and pathogenesis. Bioinformatic analysis indicates that both the ORF3 and M proteins posses at least 3 C-terminal transmembrane regions. To further characterize the biological activity of these three proteins in clinical and laboratory samples, sensitive and specific antibodies are required. Thus, the antigenic regions of ORF3, M and the entire E gene were amplified by PCR and ligated into a bacterial expression vector for expression and subsequent generation of antibodies in a mouse system. The identities of the cloned genes were confirmed by sequencing before being expressed in an in vitro bacterial system. Western Blots were used to identify the expression of the 41kDa, 42kDa and 34kDa GST-tagged viral proteins which were consistent with the bioinformatically predicted protein species. Verified fusion proteins were expressed in large quantities, quantified and concentrated for in vivo antibody production. Inoculation of 9 healthy, female Balb/C mice with the purified fusion proteins yielded high titers of polyclonal antibodies. Western Blotting was once again used to validate the production of the antibodies before their specificity was quantitatively measured using a modified competition ELISA. / South Africa

Philosophiae Doctor - PhD / Human Coronavirus NL63 (HCoV-NL63) is one of seven coronaviruses (CoVs) that cause respiratory disease in the global population. The Membrane (M) and Nucleocapsid (N) proteins are part of the core CoV-structural proteins, crucial in viral replication and virion assembly. Here the expression of HCoV-NL63 M and N was characterized across multiple in vitro systems including bacterial, insect and mammalian. To detect untagged proteins in viral structural studies, anti-peptide antibodies were generated in a mouse model. Polyclonal antisera and hybridoma-secreted antibodies exhibited specific binding to their respective full length protein antigens. Anti-peptide monoclonal antibodies were successfully generated against the HCoV-NL63 M and N proteins. During CoV infection, the interaction of CoV M and N is necessary for the production of infectious virions. For the first time, co-expressed, full length HCoV-NL63 M and N were assayed for protein-protein interaction in a mammalian cell system, allowing for native protein folding and modification. M protein formed higher order homomultimers in the presence and absence of co-expressed N.

No description available.

Thesis (M. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid (N) protein may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. In the infected cell, N was the only viral protein that was phosphorylated as shown by 32P-orthophosphate labeling and Western blot analysis and with IBV specific polyclonal chicken antibody. Using pulse-labeling with 32Porthophosphate, the IBV N protein was found to be phosphorylated in the virion, as well as at all times during infection of Vero cells. One-hour pulse-chase analysis followed by immunoprecipitation of IBV N using rabbit anti-IBV N polyclonal antibody showed that the phosphate on the protein did not fall below 70% of the maximum and remained stable. The small but reproducible drop in phosphorylation could modulate the various functions of the N protein in the infected cell. Simultaneous labeling with 32Porthophosphate and 3H-leucine of infected CEK cells indicated a 3.5-fold increase in the ratio of the 32P:3H counts per minute (cpm) on the virion N protein as compared to the 32P:3H cpm ratio of the N protein from lysates at 7 h p.i. The 32P:3H cpm ratio of the N protein from virion from infected-Vero cell lysates was 10.5X more than the 32P:3H cpm ratio of the N protein obtained at 7 h p.i. It has been shown that the N proteins from the measles and rabies viruses form helical nucleocapsid-like structures when expressed in bacteria (Schoehn et al., 2001; Warnes et al., 1995). The ability of E. coli expressed IBV N protein to form helical-nucleocapsid-like structures was investigated using transmission electron microscopy. Full-length, purified histidine-tagged IBV N protein formed nucleocapsid-like structures when expressed in bacteria. Because E. coli -expressed histidine-tagged fragments of the IBV N protein did not form helical nucleocapsid-like structures, the full-length protein is probably required for assembly of these structures. The highly conserved IBV N protein was also used as a diagnostic tool in an ELISA for detecting anti-IBV antibody in chicken serum using a specialized microwave called the BIOWAVE. The BIOWAVE improves the processing time for an ELISA.

Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid (N) protein may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. In the infected cell, N was the only viral protein that was phosphorylated as shown by 32P-orthophosphate labeling and Western blot analysis and with IBV specific polyclonal chicken antibody. Using pulse-labeling with 32Porthophosphate, the IBV N protein was found to be phosphorylated in the virion, as well as at all times during infection of Vero cells. One-hour pulse-chase analysis followed by immunoprecipitation of IBV N using rabbit anti-IBV N polyclonal antibody showed that the phosphate on the protein did not fall below 70% of the maximum and remained stable. The small but reproducible drop in phosphorylation could modulate the various functions of the N protein in the infected cell. Simultaneous labeling with 32Porthophosphate and 3H-leucine of infected CEK cells indicated a 3.5-fold increase in the ratio of the 32P:3H counts per minute (cpm) on the virion N protein as compared to the 32P:3H cpm ratio of the N protein from lysates at 7 h p.i. The 32P:3H cpm ratio of the N protein from virion from infected-Vero cell lysates was 10.5X more than the 32P:3H cpm ratio of the N protein obtained at 7 h p.i. It has been shown that the N proteins from the measles and rabies viruses form helical nucleocapsid-like structures when expressed in bacteria (Schoehn et al., 2001; Warnes et al., 1995). The ability of E. coli expressed IBV N protein to form helical-nucleocapsid-like structures was investigated using transmission electron microscopy. Full-length, purified histidine-tagged IBV N protein formed nucleocapsid-like structures when expressed in bacteria. Because E. coli -expressed histidine-tagged fragments of the IBV N protein did not form helical nucleocapsid-like structures, the full-length protein is probably required for assembly of these structures. The highly conserved IBV N protein was also used as a diagnostic tool in an ELISA for detecting anti-IBV antibody in chicken serum using a specialized microwave called the BIOWAVE. The BIOWAVE improves the processing time for an ELISA.

Au cours des deux dernières décennies, il a été démontré que les maladies émergentes et réémergentes sont liées à la santé humaine, animale et environnementale. Les infections zoonotiques sont reconnues comme étant responsables d'au moins 75 % des éclosions d'agents pathogènes dans le monde. Certains virus peuvent muter et infecter un large éventail d'hôtes, qui se propagent parmi les humains et entraînent des épidémies ou des pandémies. Les chauves-souris sont connues pour être les mammifères les plus diversifiés géographiquement et le plus répandu sur Terre et peuvent être trouvées à l'intérieur structures abandonnées, ainsi que de grottes. Au Canada, il existe dix-huit espèces de chauves-souris insectivores et huit d'entre elles se perchent au Québec. Elles peuvent loger beaucoup de pathogènes pendant l'hibernation en raison de leur métabolisme distinct. Elles peuvent aussi excréter beaucoup de particules virales par différentes voies telles que la salive, les excréments et l'écholocation. Le but de ce projet de maîtrise est de caractériser les virus que les chauves-souris pourraient potentiellement transporter au Canada, en mettant l'accent sur la détection des Coronavirus. Le premier objectif est de traiter 250 échantillons (matières fécales et organes) prélevés sur des chauves-souris sauvages, puis d'utiliser d'autres techniques biomoléculaires comme le NGS pour détecter un large spectre de virus chez ces chauves-souris. Les échantillons ont été dépistés pour les Coronavirus et les Rhabdovirus à l'aide de la PCR conventionnelle. La prévalence des Coronavirus chez les chauves-souris semble actuellement être relativement faible au Canada et de nombreux facteurs, y compris le petit nombre d'échantillons prélevés jusqu'à présent, pourraient y avoir joué un rôle. Il serait important de continuer à examiner les échantillons de chauves-souris à plus grande échelle afin de caractériser pleinement les viromes que ces animaux hébergent, pour fournir un avertissement des menaces d'épidémie ou de pandémie à la première occasion. / Over the past two decades, emerging and re-emerging diseases have been shown to be interlinked between human, animal, and environmental health. Zoonotic infections are recognized to be responsible for at least 75% of pathogen outbreaks in the world. Certain viruses can mutate and infect a wide range of hosts, which spread amongst humans and lead to epidemics/pandemics. Bats are known to be the most geographically diverse and widespread mammal on Earth and can be found inside of buildings and houses or abandoned structures, as well as caves. In Canada, there are eighteen species of insectivorous bats and eight of them roost in Québec. They can also harbor plenty of pathogens during hibernation due to their distinct metabolism. They can shed plenty of pathogens through different pathways such as saliva, excreta, and echolocation. The overall goal of this master's project is to characterize the pathogens that bats could be potentially carrying in Canada, with a focus on Coronavirus detection. The first objectiveis to process 250 samples (feces and organs) collected from wild bats in the field in Canada and then using other biomolecular techniques such as NGS to detect for the presence of other bat pathogens. The samples were screened for Coronaviruses and Rhabdoviruses using conventional PCR with no positive results. The prevalence of Coronaviruses in bats currently appear to be relatively low in Canada and, many factors, including the moderately low numbers of samples collected in Canada so far, may have played a role. It will be important to continue collecting and screening bat samples on a larger scale to fully characterize the viromes that these animals harbour, to provide warning of epidemic/pandemic threats at the earliest opportunity